Modulation of Altered Immune Parameters IL-2 and TNF-α in Diabetic Animal Models: A Therapeutic Insinuation of Metformin Beyond Diabetes.

BACKGROUND: Immunomodulatory drugs target the overall immune system, hence producing numerous toxic effects on the other organs with serious health manifestations. Due to these safety concerns, there is a need to introduce or repurpose a new drug with immunomodulatory effects with good safety, efficacy, and better tolerance. Metformin, a standard antidiabetic drug, was evaluated for its immunomodulatory effects in diabetic models in the current study. METHODOLOGY: The diabetic model was developed by intraperitoneal (IP) administration of streptozotocin (60 mg/kg). The experimental rats were divided into six groups (three diabetic and three non-diabetic) with six rats in each group. Metformin (50 mg/kg and 80 mg/kg) was given orally to both diabetic and non-diabetic groups, once a day, for 42 days. Immunomodulatory cytokines interleukin (IL)-2, IL-4, IL-5, tumor necrosis factor (TNF)-α, and interferon gamma (INF-É£) were analyzed from blood samples by BD FCAP flow cytometer. RESULTS: The results revealed a significant (p=0.002) decrease in IL-2 and TNF-α in diabetic groups in comparison to control rats. However, no significant changes were observed in IL-4, IL-5, and INF-É£ levels. Importantly, the treatment of metformin at both doses, i.e., 50 and 80 mg/kg, significantly reduced the elevated levels of IL-2 and TNF-α when compared to untreated diabetic groups. CONCLUSION: Metformin may be considered as an optimum drug candidate to reduce pro-inflammatory cytokines, IL-2 and TNF-α, that can lead to the reduction of long-term diabetic complications.

Rutin coated gold nanoparticles prevent rhabdomyolysis-induced kidney injury via down-regulation of NF-kB, iNOS, IL-6 and up-regulation of HO-1 and Kim-1 genes in mice.

The aim of this study was to evaluate the protective activity of rutin, and its gold nanoparticles (Ru-AuNPs) in rhabdomyolysis-induced acute kidney injury (AKI) model in mice. Rutin (25 and 50 mg/kg) and Ru-AuNPs (15 and 25 mg/kg) were administered to the animals for four (4) days with water deprivation for 24 hours followed by 50% glycerol injection at the dose of 10 ml/kg intramuscularly. On the next day, animals were dissected and blood and kidneys were collected. Biochemical investigations were performed to evaluate kidney functions, histological studies were carried out to see the changes at tissue level and real-time RT-PCR studies for nuclear factor-κB p50, NFκB; inducible nitric oxide synthase, iNOS; heme oxygenase-1, HO-1; interleukin-6, IL-6; and kidney injury molecule-1, Kim-1 were performed to elucidate the molecular mechanisms. Blood urea and creatinine were found to be decreased in animals treated with rutin and Ru-AuNPs. Down regulation of the mRNA expressions of iNOS, IL-6 and NFkB p50 and up-regulation of Kim-1 and HO-1 genes were observed. The efficacy of Ru-AuNPs was better than rutin alone even at a dose far less than the compound. Rutin and Ru-AuNPs alleviates kidney injury and inflammation in rhabdomyolysis-induced AKI model via anti-inflammatory and anti-oxidant pathways which make it a plausible compound for future studies.

Eugenol and liposome-based nanocarriers loaded with eugenol protect against anxiolytic disorder via down regulation of neurokinin-1 receptors in mice.

Anxiety disorder is a psychiatric disorder characterized by extreme fear or worry. It is highly prevalent worldwide which affects daily life and is also an enormous health burden. Neurokinin 1 receptor (NK1R) is a G protein coupled receptor, expressed in both central and peripheral nervous system, involved in affective behaviors. NK1R has established role in anxiety and it is also an important target for pathogenesis of anxiety disorder. Therefore, it has been hypothesized in previous studies that the blockades of NK1R may have antidepressant and anxiolytic effects. The present study deals with the molecular mechanism of protective activity of eugenol against anxiolytic disorder. A pre-clinical animal study was performed on 42 BALB/c mice. Animals were given stress through conventional restrain model. The mRNA expression of NK1R was analyzed by real time RT-PCR. Moreover, the NK1R protein expression was also examined by immunohistochemistry in whole brain and mean density was calculated. The mRNA and protein expressions were found to be increased in animals given anxiety as compared to the normal control. Whereas, the expressions were decreased in the animals treated with eugenol and its liposome-based nanocarriers in a dose dependent manner. However, the results were better in animals treated with nanocarriers as compared to the compound alone. It is concluded that the eugenol and its liposome-based nanocarriers exert anxiolytic activity by down-regulating NK1R protein expression in mice.

Frequency and antibiogram of multi-drug resistant pseudomonas aeruginosa in a Tertiary Care Hospital of Pakistan.

OBJECTIVE: To determine pathogen burden and susceptibility pattern of multi-drug resistant (MDR) Pseudomonas aeruginosa isolates from clinical specimens in Karachi. METHODS: It was In-vitro Clinical study, conducted in department of Pharmacology, Ziauddin University, and isolates were collected from various specimens such as pus, tracheal aspiration, wound swab, blood and urine in Microbiology department of Ziauddin Hospital, Nazimabad campus, Karachi. The antibiotic susceptibility pattern was determined by Kirby Bauer Disc diffusion method. Samples were processed as per procedures defined by Clinical and Laboratory Standards Institute (CLSI) guidelines 2018. RESULTS: About 55% were found to be multi drug resistant P. aeruginosa. Majority of the isolates (35.4%) were recovered from the age range 60-80 years. Maximum number of MDR P. aeruginosa was isolated from pus (33.1%) followed by tracheal aspiration (20.6%). Highest sensitivity was seen by colistin (100%) followed by ceftolozane/tazobactam (60%). Least sensitivity was observed with imipenem (19%). However, increase trend of resistance was seen among all antipesudomonal drugs. CONCLUSION: Increasing frequency of infections due to MDR P. aeruginosa is an emerging threat in our set up which can be prevented by prescribing antibiotics judiciously. Consistent lab detection and surveillance regarding this resistant pathogen is compulsory for providing effective health care to community.

Bax, Bcl-2, and Bax/Bcl-2 as prognostic markers in acute myeloid leukemia: are we ready for Bcl-2-directed therapy?

PURPOSE: Many anticancer drugs induce apoptosis in malignant cells, and resistance to apoptosis could lead to suboptimal or no therapeutic benefit. Two cytoplasmic proteins, B-cell lymphoma protein 2 (Bcl-2)-associated X (Bax) and Bcl-2, act as a promoter and an inhibitor of apoptosis, respectively. Both Bax and Bcl-2 as well as their ratio have been regarded as prognostic markers in various cancers. However, conflicting results have been reported. A clear understanding of apoptosis has also become crucial due to reports about anti-Bcl-2 chemotherapy. We explored the relationship of Bax and Bcl-2 gene expression and their ratio with the therapeutic response in acute myeloid leukemia (AML) patients. PATIENTS AND METHODS: Bone marrow and/or blood samples from 90 AML patients treated with cytarabine and daunorubicin were included. Expression of Bax and Bcl-2 was determined through real-time polymerase chain reaction by using ΔΔCt method of relative expression. RESULTS: Bax and Bcl-2 expression among marrow and blood samples correlated with each other (rs=0.5, p<0.01). Although bone marrow expression of Bax and Bcl-2 tended to remain higher among responders (median 1.01 and 0.29, respectively) as compared to non-responders (median 0.66 and 0.24, respectively), the difference failed to reach statistical significance (U=784.5 and 733; p=0.68 and 0.28, respectively). Conversely, Bax/Bcl-2 ratio was higher among poor responders (median 3.07 vs 1.78), though again failed to reach statistical significance (U=698.5, p=0.07). CONCLUSION: Expression of Bax and Bcl-2 does not differ significantly among AML patients treated with cytarabine and daunorubicin in terms of remission, relapse, resistance, overall survival, and disease-free survival, thus questioning the utility of emerging anti-Bcl-2 therapy.

MiR-16: A novel hereditary marker in breast cancer and their offspring.

OBJECTIVE: To analyse micro ribonucleic acid-16 in sera of invasive intraductal breast carcinoma in stage III and compare its expression in their daughters and healthy women. METHODS: The study took place from January 2013 till December 2015. This case-control study was conducted at the Ziauddin Cancer Hospital, Karachi, and comprised breast cancer patients and healthy individuals. Stage III invasive intraductal breast cancer patients (cases), their age-matched healthy individuals (control group A) and patients' daughters (control group B) were included. Subjects with stage I cancer and their daughters and subjects with stage IV and their daughters were also included. Serum tests were run on real-time quantitative reverse transcription polymerase chain reaction. Threshold cycle was determined and fold change was calculated. Fold change was applied between the groups. SPSS 20 was used for data analysis. RESULTS: Of the 194 participants, there were 50(25.8%) cases, 50(25.8%) group A controls, 35(18%) group B controls, 20(10.3%) stage I patients, 11(5.7%) daughters of stage 1 patients, 20(10.3%) patients of stage IV and 8(4.1%) daughters of stage IV patients. Micro ribonucleic acid-16 was higher in cases than controls (p=0.001). Group B showed significant gene expression than group A (p=0.001). Stage IV patients and daughters showed expression of micro ribonucleic acid-16 (p=0.001). Triple negative receptor cases showed a greater expression of gene (p=0.001). CONCLUSIONS: Micro ribonucleic acid-16 can be used as a prognostic, diagnostic as well as a predictive marker in breast cancer patients and their offspring.

Investigative and extrapolative role of microRNAs' genetic expression in breast carcinoma.

MicroRNAs (miRs) are non-coding ribonucleic acids consisting of about 18-22 nucleotide bases. Expression of several miRs can be altered in breast carcinomas in comparison to healthy breast tissue, or between various subtypes of breast cancer. These are regulated as either oncogene or tumor suppressors, this shows that their expression is misrepresented in cancers. Some miRs are specifically associated with breast cancer and are affected by cancer-restricted signaling pathways e.g. downstream of estrogen receptor-α or HER2/neu. Connection of multiple miRs with breast cancer, and the fact that most of these post transcript structures may transform complex functional networks of mRNAs, identify them as potential investigative, extrapolative and predictive tumor markers, as well as possible targets for treatment. Investigative tools that are currently available are RNA-based molecular techniques. An additional advantage related to miRs in oncology is that they are remarkably stable and are notably detectable in serum and plasma. Literature search was performed by using database of PubMed, the keywords used were microRNA (52 searches) AND breast cancer (169 searches). PERN was used by database of Bahria University, this included literature and articles from international sources; 2 articles from Pakistan on this topic were consulted (one in international journal and one in a local journal). Of these, 49 articles were shortlisted which discussed relation of microRNA genetic expression in breast cancer. These articles were consulted for this review.

Diagnostic, prognostic and predictive value of MicroRNA-21 in breast cancer patients, their daughters and healthy individuals.

MicroRNA-21 (miR-21) located on 17q23.1 expressed in breast cancer has anti-apoptotic ability and causes tumor cell growth. It is also involved in functions such as signal transduction pathways effecting normal cell growth and differentiation. The primary objective of the study was to identify presence of miR-21 in the serum levels of stage III invasive ductal carcinoma patients and compare its expression with age matched healthy individuals and daughters of index cases. The secondary objective was to evaluate the significance of serum miR-21 gene expression with histologically proven estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) proteins. A total of 132 subjects were recruited: 50 (cases) of stage III invasive ductal carcinoma patients who had not undergone any chemotherapy or surgery were randomly picked with exclusion of females with other types of breast carcinoma. Age-matched, 50 healthy individuals (control A) were selected by purposive sampling after confirmation of no palpable lump/s in their breasts together with 32 daughters of index cases (control B). Serum tests were run on Real Time quantitative Reverse Transcription PCR, threshold cycle was determined and fold change calculated.Normality of continuous variables was assessed by Shapiro-Wilk's test, groups compared by student t-test, Mann-Whitney test and Fisher exact test, P-value ≤ 0.05 was considered significant. We observed that miR-21 was significantly higher in cases as compared to control A and B (P = 0.001) however control B showed significant gene expression as compared to control A (P = 0.001). The cases were also divided as positive or negative for ER, PR and HER2. High expression of miR-21 in females with stage III invasive ductal carcinoma had been calculated as compared to its age matched healthy subjects. It was observed that triple negative cases showed a greater expression of gene as compared to other groups (P = 0.001). Expression of miR-21 in daughters of the cases was significantly higher as compared to healthy controls but lesser than females with invasive intraductal carcinoma. This result strengthens the concept of inheritability of disease with prediction of miR-21 as a potentially strong diagnostic and prognostic biomarker of breast cancer.

Imipenem Resistant Pseudomonas aeruginosa: The fall of the final quarterback.

OBJECTIVE: To isolate, determine the frequency, and study the demographic trends of MBL positive Pseudomonas aeruginosa from imipenem resistant isolates collected from clinical samples in a tertiary care hospital of Pakistan. METHODS: In this cross sectional study a total of 230 strains of Pseudomonas were isolated from various clinical specimens on the basis of culture and biochemical tests. Imipenem resistant isolates were selected by Kirby Bauer Diffusion technique, followed by screening for MBL production by Imipenem EDTA Combined Disk Test. Demographic details of each patient were recorded on a separate questionnaire. Chi-Square goodness-of-fit test was computed to review the isolation of MBL positive isolates (P-value ≤ 0.05) in different specimen. RESULTS: Out of 230 strains of P. aeruginosa 49.5% were imipenem resistant; MBL production was confirmed in 64.9% of the resistant isolates. Resistance to polymyxin B (12.5%) was notable. Majority of the MBL positive strains were isolated from patients aged between 20-39 years (45.9%) and the predominant source was pus (43.24%) which was found to be statistically significant (P-value=0.04). Outpatient departments (24.3%) and burn unit (21.6%) were the major places for resistant isolates. CONCLUSION: MBL production is one of the major causes of IRPA. Increasing resistance to polymyxin B is grave. Due to acquisition of MBL strains MDR P. aeruginosa has become endemic in tertiary setups.

Association of the use of bacterial cell wall synthesis Inhibitor drugs in early childhood with the Developmental Defects of Enamel.

OBJECTIVE: Our objective of the study was to determine the association between frequent use of Penicillins and Cephalosporins with developmental defects of enamel in pediatric age group. METHODS: This is a cross sectional study, conducted at Ziauddin University. A total of 367 children, having the history of either Penicillin or Cephalosporin exposure were included. The parents of children were asked to complete a questionnaire related to disease and drug history. Dental examination was carried out to assess the hypomineralization in tooth enamel based on modified Developmental Defects of Enamel (DDE) index. RESULTS: Out of 367 children, 124 (34%) were males and females were 243(66%). In the study group 22.6% (n= 83) of children were found to be hypomineralized. The maximum type of teeth defects were diffused opacities that was 12.0% (n=44). The statistically significant association (p-value < 0.05) was found between frequency of antibiotic use and hypomineralization for most teeth. Children who were exposed to either Penicillins or Cephalosporin in early childhood showed significant (p-value < 0.002) hypomineralized enamel. CONCLUSION: This study concludes that frequent use of antibiotics such as penicillins and cephalosporins has positive association with enamel hypomineralization in developing tooth structure.

Efficacy of vancomycin versus linezolid against coagulase-negative staphylococci in various clinical specimens.

BACKGROUND: Worldwide increase in antibiotic resistance has become one of the major problems. Optimal and rationale use of antibiotic is important to prevent resistance against most of the bacteria including Coagulase-negative Staphylococci (CoNS), which has now been recognized as an important pathogen for nosocomial infections. This study was carried out to determine efficacy of vancomycin and linezolid against CoNS in various clinical specimens. METHODS: A total of 2989 specimens of blood, pus and wound swab were collected from wards, casualty, intensive care units (ICU) and out-patient department (O.P.D), out of these, Staphylococci were isolated in 1017 specimens, of which 381 were identified as CoNS. Culture, gram stain, catalase, coagulase test and antimicrobial susceptibility pattern were done on these specimens according to clinical manual of microbiology. A total of thirteen most commonly used antibiotics were used in this study. Susceptibility testing was done by Kirby Bauer disc diffusion technique. RESULTS: Antimicrobial resistance of these isolates were Amoxicillin (74.8%), Amoxicillin+clavulanate (32.8%), Ciprofloxacin (35.2%), Ofloxacin (33.6%), Ceftriaxone (30.4%), Erythromycin (58.3%), Clindamycin (16.3%), Kanamycin (52.2%) Fusidic acid (41.7%), Doxycycline (24.7%), Vancomycin (2.6%) and Linezolid (0.8%) respectively. Isolates obtained from blood were 45.9%. CONCLUSION: Vancomycin showed resistance against CoNS which is a real threat for currently applied therapy against methicilin resistant CoNS. However, linezolid efficacy is higher than vancomycin against CoNS in our study, which suggests that this drug may be considered superior to vancomycin for the treatment of infections associated with CoNS.

Streptozotocin-induced insulin deficiency leads to development of behavioral deficits in rats.

Diabetes mellitus is one of the most common serious metabolic disorders in humans that develops due to diminished production of insulin (type I) or resistance to its effect (type II and gestational). The present study was designed to determine the neuropsychological deficits produced following streptozotocin-induced diabetes in rats. Rats were made diabetic by the intra-peritoneal administration of 60 mg/kg streptozotocin (STZ) which induces type-1 diabetes by the destruction "ß-cells" of pancreas. Body weight, food and water intake was monitored daily. Open field test (OFT) model, forced swim test (FST) and Morris water maze (MWM) model were performed for the evaluation of ambulation, depression-like symptoms and memory effects, respectively. After 10 days of diabetes induction the exploratory activity of rats was monitored by OFT while depression-like symptoms and memory effects in rats were analyzed by FST and MWM. Results showed that there was no significant effect of STZ-induced diabetes on body weight but food and water intake of STZ-induced diabetic rats was significantly increased. Exploratory activity was significantly decreased and short-term and long-term memory was significantly impaired while the depression-like symptoms was significantly increased in STZ diabetic rats. Thus, it may be suggested that STZ-induced diabetes alters the brain functions and may play an important role in the pathophysiology of certain behavioral deficits like depression, impaired learning and memory functions related to diabetes. This finding may be of relevance in the pathophysiology and in the clinical picture, which could be related to an altered brain serotonin metabolism and neurotransmission and may possibly be related to neuropsychiatric disorders in diabetic patients.

Influence of serotonergic 5-HT2C receptor antagonist mesulergine in the reversal of memory deficits induced by mCPP.

OBJECTIVE: To determine the effect of non-selective 5-HT2C antagonist mesulergine and 5-HT2C agonist mCPP (metachlorophenylpiperazine) on learning acquisition (LA), short-term memory (STM) and long-term memory (LTM). STUDY DESIGN: Experimental study. PLACE AND DURATION OF STUDY: Department of Biochemistry, University of Karachi, from December 2009 to June 2010. METHODOLOGY: Twenty-four male albino Wistar rats were used in this study. The agonist and antagonist (mCPP and mesulergine) were injected intraperitoneally at a dose 3.0 mg/kg in volumes of 1 ml/kg. Control animals were injected with saline (1 ml/kg). Animals were randomly divided into four groups (n=6). 1st being control group, 2nd being mCPP injected group, 3rd being mesulergine injected group and 4th group being injected with both mesulergine and mCPP. Behavioural activities of rats were monitored after 30 minutes of injection. For assessment of memory functions, water maze apparatus was used. RESULTS: Administration of mCPP impaired STM, LTM and LA of rats. Mesulergine injected rats exhibited no alteration in memory functions. However, when it was injected with mCPP then there were no memory deficits induced by mCPP. CONCLUSION: Ability of 5-HT2C receptor antagonist mesulergine to block the memory impairment effect of mCPP indicated an important regulatory role of 5-HT2C receptors in cognitive processes.

Drug-prescribing patterns during pregnancy in the tertiary care hospitals of Pakistan: a cross sectional study.

BACKGROUND: The rationale for use of drugs during pregnancy requires a careful assessment as in addition to the mother, the health and life of her unborn child is also at stake. Information on the use of drugs during pregnancy is not available in Pakistan. The aim of this study was to evaluate the patterns of drug prescriptions to pregnant women in tertiary care hospitals of Pakistan. METHODS: This was a cross-sectional study conducted at five tertiary care hospitals of Pakistan. Copies of outpatient medicinal prescriptions given to pregnant patients attending the antenatal clinics were collected. The drugs were classified according to the pharmacological class and their teratogenic potential. RESULTS: All the pregnant women attending the antenatal clinics received a prescription containing at least one drug. A total of 3769 distinct prescriptions given to different women were collected. Majority of the women who received the prescriptions belonged to third trimester (55.4%) followed by second (33.6%) and first trimester (11.0%). On an average, each prescription contained 1.66 +/- 0.14 drugs. The obstetricians at Civil Hospital, Karachi and Chandka Medical College Hospital, Larkana showed a tendency of prescribing lesser number of drugs compared to those in other hospitals. Anti-anemic drugs including iron preparations and vitamin and mineral supplements (79.4%) were the most frequently prescribed drugs followed by analgesics (6.2%) and anti-bacterials (2.2%). 739 women (19.6%) received prescriptions containing drugs other than vitamin or mineral supplements. Only 1275 (21.6%) of all the prescribed drugs (n = 6100) were outside this vitamin/mineral supplement class. Out of these 1275 drugs, 29 (2.3%) drugs were prescribed which are considered to be teratogenic. Misoprostol was the most frequently prescribed (n = 6) among the teratogenic drugs followed by carbimazole (n = 5) and methotrexate (n = 5). Twenty nine pregnant women (0.8% of all the women studied) were prescribed these teratogenic drugs. CONCLUSION: Less than one percent of the pregnant women attending tertiary care hospitals in Pakistan are prescribed teratogenic drugs. The prescribing practices of Pakistani physicians are similar to those in western countries.